View field expansion method based on Airy-like beam and detection objective lens depth-of-field enhancement

Abstract

The invention discloses a view field expansion method based on Airy-like light beams and depth-of-field enhancement of a detection objective lens. The method comprises the following steps: shaping and filtering light beams emitted by a light source into Airy-like light beams; the light beam forms an illumination light sheet through the scanning galvanometer; the illumination light sheet is focused at the focus of the excitation objective lens to excite a sample in the sample pool to generate a fluorescence signal; the detection objective lens is combined with the conical lens and the barrel lens to collect fluorescence signals of the focal plane, stray light is filtered out through the optical filter, and then imaging is carried out on the camera. The scattering absorption effect of biological tissues is effectively weakened, and the method is more suitable for large-volume biological sample imaging; and the imaging depth of the detection light path can be obviously increased.

Description

Technical Field

[0001] The present invention relates to the field of bio-optical microscopic imaging technology, and in particular to a method for expanding the field of view based on an Airy-like beam and a depth-of-field enhancement of a detection objective lens. In a light-sheet fluorescence microscope, an Airy-like illumination beam and a depth-of-field enhancement of a detection objective lens (an axicon lens and a cubic phase mask perform wavefront encoding on a detection light path) are applied to achieve imaging field enhancement. Background Art

[0002] Optical imaging technology has long been an important tool in biomedical research, allowing researchers to effectively observe the components of organisms and cells and their dynamic processes. Among them, the light sheet fluorescence microscopy method has the characteristics of high temporal resolution, low phototoxicity and photobleaching due to its special illumination and detection optical path design. At the same time, it can also take into account the imaging spatial resolution and imaging depth, allowing researchers to observe the life activities of living samples for a long time, which is of great significance to life science research.

[0003] The cross-sectional radius of the Gaussian beam used in traditional light sheet microscopy shows the characteristics of a hyperbola along the propagation direction, and its Rayleigh length is relatively short, resulting in a very limited field of view of the Gaussian light sheet fluorescence microscope: at the waist of the Gaussian beam, the axial resolution of the imaging is the best; once it exceeds the Rayleigh range, the diffraction effect of the Gaussian beam will quickly appear, and its resolution ability will be significantly reduced, showing that the Gaussian light sheet has axial resolution and field of view constraints during imaging. In addition, living biological samples are often opaque and have strong absorption and scattering effects on light. Gaussian light sheets are easily affected by the above-mentioned photon-tissue interaction, resulting in reduced imaging quality of deep tissues.

[0004] Non-diffracting light beams, especially Airy beams, are often used to improve the imaging field of view of light sheet fluorescence microscopes. Airy beams have low spatial coherence and are less affected by sample inhomogeneities. They have high contrast and uniform resolution over a wider field of view, thus enabling a longer imaging field of view. However, Airy light sheet microscopes also have shortcomings. Their self-bending characteristics cause the main lobe and side lobes of the Airy beam on both sides of the field of view to exceed the depth of field of the detection objective, affecting the collection of fluorescence signals on both sides. During the imaging process, it is difficult to achieve higher resolution and signal-to-noise ratio, thus limiting the imaging field of view of the Airy light sheet microscope imaging system.

[0005] The most classic way to increase the depth of field is to reduce the aperture of the objective lens. Increasing the numerical aperture of the objective lens can reduce the diameter of the Gaussian beam waist, and the Rayleigh distance will decrease accordingly. The point spread function (PSF) of an ordinary imaging system is Gaussian. The numerical aperture of the detection objective lens can be reduced by reducing the size of the aperture diaphragm, thereby achieving the purpose of increasing the depth of field. As the aperture of the objective lens decreases, the depth of field of the imaging system gradually increases, but the reduction of the pupil will reduce the transmission of the light signal, which is not conducive to the collection of fluorescence signals, resulting in poor contrast of the imaging results.

[0006] In addition, wavefront coding is an optical method commonly used to extend the depth of field of incoherent imaging systems. By introducing an optimized phase mask on the pupil plane, the optical transfer function (OTF) of the system is modulated to make it defocus invariant. The cubic phase mask is the most primitive phase mask introduced by the fixed phase method. By using wavefront coding technology to extend the depth of field of the detection optical element in the light sheet microscope, the light sheet can be accommodated anywhere within the extended axial range, thereby enabling rapid scanning of the light sheet on the sample. Therefore, the mechanical displacement of the stage is not required during the imaging process. Large-volume imaging can be performed by only axially scanning the light sheet, which reduces the artifacts caused by mechanical displacement and increases the depth of field range of the detection objective.

[0007] However, wavefront coding also has certain limitations. In order to achieve the best imaging effect, the parameters of the phase mask need to be optimized, which is not simple and easy. At the same time, the manufacturing process of the phase mask is complicated and the customization price is high. Summary of the invention

[0008] The present invention provides a method for expanding the field of view based on an Airy-like beam and a depth of field enhancement of a detection objective lens. The present invention effectively reduces the scattering and absorption effects of biological tissues and is more suitable for imaging large-volume biological samples; and can significantly increase the imaging depth of the detection optical path, as described below:

[0009] A method for extending the field of view based on an Airy-like beam and depth of field enhancement of a detection objective lens, the method comprising:

[0010] The light beam emitted by the light source is shaped and filtered into an Airy-like beam;

[0011] The light beam passes through the scanning galvanometer to form an illumination light sheet; the illumination light sheet is focused at the focus of the excitation objective lens, exciting the sample in the sample pool to generate a fluorescence signal;

[0012] The detection objective lens is combined with the conical lens and the tube lens to collect the fluorescence signal in the focal plane, and the image is formed on the camera after the stray light is filtered out by the filter.

[0013] The step of shaping and filtering the light beam emitted by the light source into a quasi-Airy beam is as follows:

[0014] Changing the power of the phase changes the self-bending degree of the generated beam. The phase formula after modulation is:

[0015] P Airy-like (u,v)=R(u,v)exp[2pαi[sign(u,v)(u 2 +v 2 )+(u 3 +v 3 )+…]]

[0016] The beam generated by this power superposition is called an Airy-like beam. Airy-like (u,v) is the phase of the modulated Airy beam, R(u,v) is the pupil aperture function, α is the scale factor of the Airy beam, i is the imaginary unit, and u,v are normalized coordinates.

[0017] Wherein, the light beam forms an illumination light sheet through a scanning galvanometer:

[0018] The aconic lens is placed between the objective lens and the tube lens. A point light source is placed at different positions z in front of the objective lens. The point light source is diffracted and imaged on the camera. The axial PSF of the detection light path is obtained by moving the axial z position of the point light source.

[0019] The PSF of the detection light path after adding the aconic lens presents a Bessel shape. At the position z = 0 μm, the PSF gathers into a ring shape. The position z = -620 μm where the PSF energy of the modified detection light path is the highest is defined as the focal position of the modified light path.

[0020] By controlling variables, a conical lens with a base angle of 1° or 0.5° is selected for imaging; a plano-convex conical lens is used for imaging to obtain stronger light intensity and lower cost; the axial intensity distribution of the PSF of the detection light path is changed by changing the aperture size of the conical lens, and a conical lens with a larger aperture is selected; and the optical axes of the conical lens and the objective lens are ensured to coincide.

[0021] The PSF of the detection light path using the three-dimensional phase mask presents an Airy shape:

[0022] Place the cubic phase mask between the detection objective lens and the tube lens, place a point light source at different positions z in front of the objective lens, diffract the point light source, and image it on the camera. By moving the axial z position of the point light source, the axial PSF and MTF images of the detection light path are obtained.

[0023] The PSF of the detection optical path after adding the three-time phase mask is Airy distributed. The MTF curve of the modified optical path remains flat within the distance of z=0μm~15μm, and a certain information transmission capability can be maintained within this distance.

[0024] By controlling the variables, the three-dimensional phase mask is placed behind the objective lens. 1 = 0mm position to produce the best wavefront modulation effect; changing the scale factor of the phase mask can increase the range of its depth of field modulation. However, due to the self-bending characteristics of the Airy-shaped PSF, the imaging results on both sides of the focal plane will be displaced, which will have a great impact on the subsequent image processing.

[0025] The beneficial effects of the technical solution provided by the present invention are:

[0026] 1) Compared with the conventional Airy light sheet, the Airy light sheet of the present invention can obtain a larger imaging field of view of 1220 μm while maintaining a certain resolution, which is 7% and 5% larger than the Airy light sheet with NA=0.175 and α=25 of the same axial resolution; the Airy light sheet with extended depth of field can obtain an imaging field of view of 490 μm, which is about 36% higher than the field of view of the Airy light sheet; the Airy light sheet with extended depth of field can obtain an imaging field of view of 1300 μm, which is about 46% higher than the field of view of the Airy light sheet;

[0027] 2) The intensity of the Airy light sheet of the present invention is focused on both sides of the field of view, which can effectively reduce the scattering and absorption effects of biological tissues and is more suitable for imaging large-volume biological samples;

[0028] 3) The present invention can significantly increase the imaging depth of the detection optical path. Under the basic parameter conditions set by the present invention, the depth of field of the detection optical path can be increased to more than 1000 μm, which is 50 times greater than that of the ordinary detection optical path;

[0029] 4) The present invention has experimentally demonstrated that the field-enhanced Airy light sheet microscopy system can provide a new method for the treatment of traumatic brain injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A principle flow chart of the light sheet field of view expansion microscopic imaging system provided by the present invention;

[0031] Figure 2 A light path diagram of the light sheet field-of-view expansion microscopic imaging system provided by the present invention;

[0032] Figure 3 Schematic diagram of the Airy-like light sheet phase diagram, PSF and MTF images along the x-axis with different power superposition;

[0033] Among them, (ah) are the phase images (i), PSF (ii) and MTF images (iii) of the Airy-like light sheet with different superposition powers; the white dotted line indicates the frequency threshold of 3.8%.

[0034] Figure 4 A schematic diagram comparing Airy light sheets and Airy light-like sheets;

[0035] Among them, (a) is the PSF image of the Airy light sheet along the x-axis; (b) is the MTF image of the Airy light sheet; (c) is the PSF image of the quasi-Airy light sheet along the x-axis; (d) is the MTF image of the quasi-Airy light sheet; (e) is the intensity distribution at x=0μm; (f) is the MTF curve at x=0μm and x=300μm; the white dotted line indicates the frequency threshold of 3.8%.

[0036] Figure 5 Schematic diagram of the axial PSF and MTF images of the detection light path before and after adding the axicon;

[0037] Figure 6 A schematic diagram of the image simulation results at different positions using the USFA resolution test chart before and after adding the aconic lens modification;

[0038] Among them, (a) is the simulated imaging result when the detection light path is not modified; (b) is the enlarged image of Figure (a); (c) is the deconvolution imaging result after the detection light path is modified; (d) is the large image of Figure (c); (i-vi) are schematic diagrams of the imaging results at different axial positions.

[0039] Figure 7 Schematic diagram of the change of PSF of the detection optical path before and after adding three phase masks;

[0040] Among them, (a) is the PSF of the detection light path before the transformation; (b) is the MTF of the detection light path before the transformation; (c) is the PSF of the detection light path after the transformation; (d) is the MTF of the detection light path after the transformation; (e) is the x-axis intensity distribution when z=0μm; (f) is the MTF curve at different positions.

[0041] Figure 8 A schematic diagram of the image simulation results at different positions using the USFA resolution test chart before and after adding the three-time phase mask modification;

[0042] Among them, (a) is the simulated imaging result when the detection light path is not modified; (b) is the enlarged image in Figure (a); (c) is the deconvolution imaging result after the detection light path is modified; (d) is the enlarged image in Figure (c); (i-vi) are schematic diagrams of imaging results at different axial positions.

[0043] Fig. 9 Schematic diagram of the actual PSF of the combination of the Airy light sheet illumination optical path PSF and the depth of field extended detection optical path.

[0044] Among them, (a, d) are the Airy-like excitation light sheet PSF; (b) is the detection light path PSF without depth of field extension; (c) is the actual PSF of the light sheet microscopy system without depth of field extension; (e) is the detection light path PSF with depth of field extension; (f) is the actual PSF of the light sheet microscopy system with depth of field extension; (g) is the MTF image of the light sheet microscopy system without depth of field extension; (h) is the MTF image of the light sheet microscopy system with depth of field extension.

[0045] In the attached figure, the list of components is as follows:

[0046] 1: light source; 2: first λ / 2 half-wave plate;

[0047] 3: Polarization beam splitter; 4: Aperture;

[0048] 5: second λ / 2 half-wave plate; 6: first lens;

[0049] 7: Pinhole filter; 8: Second lens;

[0050] 9: spatial light modulator; 10: third lens;

[0051] 11: the fourth lens; 12: scanning galvanometer;

[0052] 13: fifth lens; 14: sixth lens;

[0053] 15: Excitation objective lens; 16: Detection objective lens;

[0054] 17: Axis lens; 18: Tube lens;

[0055] 19: filter; 20: camera. DETAILED DESCRIPTION

[0056] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention are described in further detail below.

[0057] Airy beams are widely used in the illumination optical path of light sheet microscopy systems due to their non-diffraction characteristics. Compared with Gaussian beams, they can obtain a larger imaging field of view while maintaining a certain axial resolution, and have the advantages of high resolution and fast imaging. However, due to the self-bending characteristics of the Airy beam, the light sheet generated by it is limited by the depth of field of the detection objective and cannot maximize the potential of the imaging field of view. The embodiments of the present invention further release the single-scan field of view potential of Airy light sheet microscopy from two perspectives: on the one hand, based on the illumination optical path, an "Airy-like" beam is used to slow down the self-bending degree of the illumination beam, so that a larger proportion of it is within the depth of field limit, thereby achieving an increase in the field of view; on the other hand, the depth of field of the detection objective is increased, and the detection optical path is wavefront encoded using an axicon lens and a cubic phase mask, so as to collect more fluorescence signals on both sides of the field of view, and ultimately increase the field of view of the Airy light sheet microscopy system.

[0058] The field-enhanced Airy light sheet microscopy system can achieve single-shot image capture of a larger range, meeting the higher imaging requirements of modern biology. It can be used for pathological analysis of disease models and exploration of physiological mechanisms, and can provide new methods for the treatment of diseases such as traumatic brain injury.

[0059] In view of the problems of the above-mentioned light sheet imaging technology in the actual biological tissue imaging process, that is, most biological samples are highly scattering and absorbing media composed of water and fat, which have strong scattering and absorption effects on light, which causes serious energy attenuation of the light sheet during the propagation of the sample, making the light sheet imaging limited in the depth of the sample. Due to the rapid diffraction of Gaussian beams, the axial resolution and field of view of traditional light sheet microscopy technology are contradictory when imaging samples, resulting in poor axial resolution at the edge of the imaging field of view, which is not suitable for large field of view imaging. At the same time, although the reduction of the numerical aperture of the objective lens can gradually increase the depth of field of the imaging system, it will reduce the transmission of the light signal, which is not conducive to the collection of fluorescence signals, resulting in poor contrast of the imaging results. However, there are problems such as difficulty in parameter optimization of phase masks.

[0060] An embodiment of the present invention provides a light sheet microscopy system with an expanded field of view. Based on the illumination optical path, the system uses an "Airy-like" beam to slow down the self-bending degree of the illumination beam, so that a larger proportion of the illumination beam is within the depth of field limit, thereby achieving an increase in the field of view; based on the imaging optical path, a conical lens and a cubic phase mask are introduced into the imaging system composed of an objective lens and a tube lens to perform wavefront encoding on the detection optical path, thereby collecting more fluorescence signals on both sides of the field of view, and ultimately increasing the field of view of the Airy light sheet microscopy system.

[0061] Example 1

[0062] The embodiment of the present invention is a method for expanding the field of view based on Airy-like beams and depth of field enhancement of a detection objective lens, the method comprising the following steps:

[0063] 101: shaping and filtering the light beam emitted by the light source into an Airy-like beam;

[0064] That is, the laser emitted by the light source is expanded through a laser beam expansion system, a spatial light modulator, a pinhole filter and other shaping devices to expand the beam diameter, filter out high-frequency components, and generate an Airy-like beam.

[0065] 102: the light beam passes through a scanning galvanometer to form an illumination light sheet;

[0066] That is, the light beam is magnified by the relay lens group so as to completely cover the entrance pupil of the excitation objective lens. The relay lens group and the scanning galvanometer form a scanning lens. The scanning frequency and amplitude and other parameters of the scanning lens are controlled by a signal generator to make the light beam scan along the y-axis to generate a light sheet.

[0067] 103: The illumination light sheet is focused at the focus of the excitation objective lens to excite the sample in the sample pool and generate a fluorescence signal;

[0068] 104: The detection objective lens is combined with the conical lens and the tube lens to collect the fluorescence signal in the focal plane, and the image is formed on the camera after the stray light is filtered out by the filter.

[0069] Example 2

[0070] 1. Generation and analysis of Airy-like beams

[0071] The wave packet of the Airy beam is a Gaussian distribution with a cubic phase factor, so the Airy beam can be generated by loading a phase pattern on a Gaussian beam and a spatial light modulator (SLM). The modulation phase formula for generating an Airy beam is:

[0072] P(u,v)=R(u,v)exp[2pαi(u 3 +v 3 )] (1)

[0073] Where R(u,v) is the aperture function, u and v are normalized coordinates, and α is the scale factor of the Airy beam. Changing the value of α can change the propagation trajectory of the Airy beam. The larger the α value, the greater the non-diffraction propagation distance of the Airy beam, but its main lobe will also become coarser, and the corresponding axial resolution will become worse.

[0074] In the experiment, the self-bending degree of the generated beam can be changed by changing the power of the phase. The phase formula after modulation is:

[0075] P Airy-like (u,v)=R(u,v)exp[2pαi[sign(u,v)(u 2 +v 2 )+(u 3 +v 3 )+…]] (2)

[0076] The beam generated by this power superposition is called an Airy-like beam. Airy-like beams can effectively slow down the self-bending of Airy beams and obtain a larger imaging field of view within a limited depth of field. Compared with the Bessel and Airy beam generation methods, the generation of Airy-like beams is faster and simpler. By using a spatial light modulator to load different phases, different illumination beams can be quickly switched, thereby comparing the advantages and disadvantages of different illumination beams.

[0077] Through P Airy-like The powers in the formula are superimposed in different ways to generate Airy-like light sheets with different propagation characteristics. In formula (2), numbers are used to concisely represent the superposition terms of different powers, where 2 represents sign(u,v)(u 2 +v 2 ), 3 represents sign(u,v)(u 3 +v 3 ), 4 represents sign(u,v)(u 4 +v 4 ), 5 represents sign(u,v)(u 5 +v 5 ), and so on.

[0078] Through simulation experiments on Airy-like light sheets with different power terms, their imaging fields of view and axial resolutions at x = 0 μm are statistically compared. On this basis, they are compared with Airy light sheets with controlled illumination numerical aperture NA and phase factor α with the same axial resolution to evaluate the increase in imaging field of view.

[0079] Through the above analysis, it can be concluded that under the same axial resolution, superimposing an Airy-like sheet with a power term of 2+3+4+5+6+4 can maximize the imaging field of view. When the axial resolution is 4.35μm, compared with the Airy sheet with a reduced NA, the imaging field of view of this type of Airy sheet can be increased by 7.0%; compared with the Airy sheet with an increased α value, the imaging field of this type of Airy sheet can be increased by 5.0%.

[0080] 2. Depth of field extension of detection objective lens based on aconic lens

[0081] In the detection optical path designed in the embodiment of the present invention, the aconic lens is placed between the objective lens and the tube lens. In the theoretical simulation, a point light source is placed at different positions (z) before the objective lens, the point light source is diffracted, and an image is formed on the camera. By moving the axial (z) position of the point light source, the axial PSF of the detection optical path can be obtained.

[0082] By comparing the axial PSF and MTF images of the ordinary detection optical path (hereinafter referred to as before modification, Before) and the detection optical path with an added conical lens (hereinafter referred to as after modification, After), it can be seen that: in the optical path before modification, the PSF presents a Gaussian shape, and the PSF on both sides of the focus of the detection objective lens diverges rapidly. The corresponding MTF shows that only a distance of 20μm can transmit information, so the depth of field of the optical path before modification is small; the PSF of the detection optical path after modification presents a Bessel shape, which can remain non-divergent over a longer distance. However, its focal position is different from that before modification. At the position z=0μm, the PSF converges into a ring shape. The position z=-620μm where the PSF energy of the detection optical path after modification is the highest is defined as the focal position of the modified optical path. The corresponding MTF of the detection optical path after modification shows that it has the ability to transmit information over a longer axial distance, so the depth of field of the optical path after modification is extended.

[0083] By controlling the variables, the influence of the position, bottom angle, shape and other factors of the aconic lens on the point spread function of the detection light path is analyzed respectively, and it can be obtained that: a) the position of the aconic lens has a weak effect on the PSF of the detection light path; b) the larger the bottom angle of the aconic lens, the closer the PSF of the detection light path is to the objective lens, and the shorter the working distance of the objective lens becomes. Therefore, in actual work, it is appropriate to select an aconic lens with an angle of 1° or 0.5° for imaging; c) the PSF of the detection light path generated by the plano-concave aconic lens and the plano-convex aconic lens are in opposite directions. The PSF generated by the plano-concave aconic lens is on the side away from the focal plane and has lower intensity. After weighing the pros and cons, in actual work, the use of a plano-convex aconic lens for imaging can obtain stronger light intensity and lower cost; d) changing the aperture size of the aconic lens can change the axial intensity distribution of the PSF of the detection light path. In actual work, an aconic lens with a larger aperture should be selected; e) the deviation of the aconic lens from the optical axis of the objective lens will produce a deformed lateral PSF and cause imaging offset. Therefore, in actual imaging, the optical axes of the aconic lens and the objective lens should be ensured to coincide.

[0084] 3. Depth of Field Extension Based on Cubic Phase Mask

[0085] The application of the cubic phase mask can change the optical transfer function of the detection light path, making it have an Airy-shaped PSF and thus have defocus invariance.

[0086] By experimentally comparing the axial PSF and MTF images of the ordinary detection optical path (hereinafter referred to as before modification, Before-3PM) and the detection optical path with a triple phase mask (Tri-Phase Mask, 3PM, proportional factor α=7) (hereinafter referred to as after modification, After-3PM), it can be seen that: the Gaussian PSF of the optical path before the modification diverges rapidly on both sides of the focus of the detection objective lens, and the divergent part is difficult to focus and image. The corresponding MTF has an axial information transmission distance of only 20μm, indicating that the depth of field of the optical path before the modification is small; the PSF of the detection optical path after the modification presents an Airy shape, and the corresponding MTF can transmit information within a longer distance, indicating that the depth of field of the optical path after the modification has been effectively increased.

[0087] By controlling the variables and analyzing the influence of the position and scale factor of the secondary phase mask on the point spread function of the detection optical path, we can obtain the following results: a) The position of the tertiary phase mask has a great influence on the increase of the depth of field of the detection objective lens. 1 = 0mm, the cubic phase mask can produce the best wavefront modulation effect; b) Changing the scale factor α of the phase mask can increase the range of its depth of field modulation. However, due to the self-bending characteristics of the Airy-shaped PSF, the imaging results on both sides of the focal plane will be displaced, which will have a great impact on the subsequent image processing.

[0088] The embodiment of the present invention belongs to the field of microscopic imaging technology, and discloses an Airy beam field of view enhancement system based on Airy-like illumination light sheet and detection objective lens depth of field enhancement. It can be targeted at different biological tissue samples and meet the needs of different biological samples for high-precision imaging at depth. The example compares the imaging method with the ordinary Gaussian light sheet, and its excellent imaging effect can be seen; it can image large samples at depth, solving the problems of the traditional light sheet microscope with a small field of view, poor imaging effect at depth, and serious reduction in axial resolution, and realizes further application in the field of light sheet microscopic imaging.

[0089] Unless otherwise specified, the models of the components in the embodiments of the present invention are not limited, and any device that can perform the above functions may be used.

[0090] Those skilled in the art will appreciate that the accompanying drawing is only a schematic diagram of a preferred embodiment, and the serial numbers of the embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for expanding the field of view based on Airy-like beams and depth of field enhancement of a detection objective lens, characterized in that: The method comprises: The light beam emitted by the light source is shaped and filtered into an Airy-like beam; The light beam passes through the scanning galvanometer to form an illumination light sheet; the illumination light sheet is focused at the focus of the excitation objective lens, exciting the sample in the sample pool to generate a fluorescence signal; The detection objective lens is combined with the conical lens and the tube lens to collect the fluorescence signal in the focal plane, and the image is formed on the camera after the stray light is filtered out by the filter.

2. The method for expanding the field of view based on Airy-like beam and depth of field enhancement of the detection objective lens according to claim 1, characterized in that: The method of shaping and filtering the light beam emitted by the light source into a quasi-Airy beam is: Changing the power of the phase changes the self-bending degree of the generated beam. The phase formula after modulation is: P Airy-like (u,v)=R(u,v)exp[2pαi[sign(u,v)(u 2 +v 2 )+(u 3 +v 3 )+…]] The beam generated by this power superposition is called an Airy-like beam. Airy-like (u,v) is the phase of the modulated Airy beam, R(u,v) is the pupil aperture function, α is the scale factor of the Airy beam, i is the imaginary unit, and u,v are normalized coordinates.

3. The method for expanding the field of view based on Airy-like beam and depth of field enhancement of the detection objective lens according to claim 1, characterized in that: The light beam forms an illumination light sheet through a scanning galvanometer: The aconic lens is placed between the objective lens and the tube lens. A point light source is placed at different positions z in front of the objective lens. The point light source is diffracted and imaged on the camera. The axial PSF of the detection light path is obtained by moving the axial z position of the point light source. The PSF of the detection light path after adding the aconic lens presents a Bessel shape. At the position z = 0 μm, the PSF gathers into a ring shape. The position z = -620 μm where the PSF energy of the modified detection light path is the highest is defined as the focal position of the modified light path. By controlling variables, a conical lens with a base angle of 1° or 0.5° is selected for imaging; a plano-convex conical lens is used for imaging to obtain stronger light intensity and lower cost; the axial intensity distribution of the PSF of the detection light path is changed by changing the aperture size of the conical lens, and a conical lens with a larger aperture is selected; and the optical axes of the conical lens and the objective lens are ensured to coincide.

4. The method for expanding the field of view based on Airy-like beam and depth of field enhancement of the detection objective lens according to claim 3, characterized in that: The PSF of the detection light path using the three-dimensional phase mask presents an Airy shape: Place the cubic phase mask between the detection objective lens and the tube lens, place a point light source at different positions z in front of the objective lens, diffract the point light source, and image it on the camera. By moving the axial z position of the point light source, the axial PSF and MTF images of the detection light path are obtained. The PSF of the detection optical path after adding the three-phase mask is Airy distributed. The MTF curve of the modified optical path remains flat within the distance of z=0μm~15μm, and a certain information transmission capability can be maintained within this distance. By controlling the variables, the three-dimensional phase mask is placed at the position d1=0mm behind the objective lens to produce the best wavefront modulation effect; changing the scale factor of the phase mask can increase the range of its depth of field modulation. However, due to the self-bending characteristics of the Airy-shaped PSF, the imaging results on both sides of the focal plane will be displaced, which has a great impact on the subsequent image processing.

Images